The bleeding time determination is an important test frequently performed before major surgery. The conventional bleeding test is performed "in vivo" (meaning, in the living body) by placing a blood pressure cuff at 40 mm Hg on a surgery patient's arm and cutting an incision of standard size into the arm. The time for the blood to form a clot, thereby stopping the flow of blood, is measured using a stop watch. Normal values are between one to six minutes. This wide range renders the test inaccurate and insensitive. Furthermore, the test necessarily causes pain and scars the patient's arm.
Blood clotting is a complicated physiological process that involves various proteins called coagulation factors, as well as cells called platelets. The platelets adhere and aggregate at the site of the wound for proper clotting. The bleeding time test measures the total effect of all the factors working in concert. Unfortunately, if the bleeding is prolonged, the in vivo test will not provide information to the clinician to isolate the deficiency which is causing the prolonged bleeding time. For example, the standard in vivo test cannot differentiate between platelets not adhering and the absence of a coagulation factor.
A blood vessel wall has several layers. The innermost layer, that which is in contact with the flowing blood, is made of endothelial cells. These cells prevent platelets from adhering. The outer layers are called the subendothelium, and contain collagen and other proteins which promote platelet adherence.
At the cut site, the vessel injury exposes collagen and other subendothelial proteins which promote platelet adhesion and aggregation. The strongest of these proteins is collagen. Collagen activates prokallikrein and this in turn initiates activation of an intrinsic coagulation pathway. There are a total of thirteen coagulation factors (F I-F XIII), where each activates the others. Platelets also interact with coagulation factors, mainly factors X, XI, and XII. Platelets release proteins from their granules, including one called platelet factor 3 (PF3) which acts as a procoagulant.
As platelets breakdown due to enzymes in granules, phopholipids on the internal side of the platelet membrane are exposed which enhances an extrinsic coagulation pathway. The thirteen different coagulation factors are divided between the intrinsic and extrinsic coagulation pathways. As more and more platelets adhere, an aggregation of platelets forms, plugging the hole in the vessel, and promoting the coagulation factors to form fibrin. That is, the end result of the two coagulation pathways is insoluble fibrin, a fibrous protein that interweaves between the aggregated platelets. The cut vessel also releases coagulation factors causing the vessel downstream of the cut to constrict.
There are two tests which measure the function of plasma coagulation factors. A first test measures the cascading extrinsic pathway and is known as the prothrombin time (abbreviated "PT"). A second test measures the intrinsic pathway and is called the partial thromboplastin time (abbreviated "PTT"). The PT and PTT tests are performed on every patient going to surgery. If one of these tests is abnormal, additional tests are conducted to look for a factor(s) deficiency. These are routine tests currently performed in the medical laboratory.
Platelet function studies are performed primarily by three routine methods: platelet aggregation, platelet adhesion, and in vivo bleeding time test. The platelet aggregation test is performed by the medical laboratory and utilizes platelet rich plasma separation from the patient's body. Aggregating agents (e.g., collagen, ADP, epinephrine, and ristocetin) are added. As the platelets aggregate, the optical density decreases with time. Aggregation studies are not exact and do not replicate the formation of a platelet plug at the site of a cut vessel.
The platelet adhesion test is measured by pushing blood through a plastic tube filled with tiny glass beads. The beads serve as foreign material to which the platelets adhere. The platelet count is measured prior to passing blood through the column and after leaving the column and reflects the percent platelets retained. Other sophisticated tests using monoclonal antibodies and chromogenic assays can also be used to measure specific membrane or granule proteins.
It would be advantageous to have an "in vitro" (meaning, outside of the living body) test that more accurately mimics what occurs at the site of a wound. Blood could be obtained by conventional blood gathering techniques and the bleeding time could then be conveniently measured without subjecting the patient to additional incisions.
U.S. Pat. No. 5,089,422, also to Dr. Daniel Brubaker, discloses an in vitro bleeding time determination apparatus. The apparatus includes a tubular device formed by heating, blowing, and stretching polyethylene tubing and a fabric membrane impregnated with clot-promoting proteins. The membrane is epoxyed onto a window in the tube. The tubular device proved effective in experimental tests, but the blown tube was not conductive to mass, low cost production. Additionally, the device experienced leakage around the epoxyed membrane. In some experimental series, the coefficient of variation was over 20%.
Another problem concerning the handling of blood is health safety. Blood samples may carry sexually transmitted infectious agents such as HIV 1 and 2, Hepatitis B, syphilis, etc. which could infect the clinician if proper safeguards are not in place.
This invention provides an in vitro bleeding time determination device that is suitable for low cost manufacturing. The device of this invention also promotes health safety in that the bleeding time tests can be conducted without handling the blood or without the risk of blood escaping the device to spill onto the clinician.
The in vitro testing device of this invention permits more standardized bleeding time tests than can be accomplished by the in vivo techniques. Variables such as depth of wound, length of wound, blood pressure of the patient, and location of the wound on the patient's body are all automatically compensated for since the test is independent of the patient's body. Bleeding time can therefore be isolated as the sole testing variable.
The device of this invention can further be used to systematically exclude specific blood clotting factors from the test, thereby testing each variable individually. The PT and PTT tests can therefore be augmented, and perhaps replaced.